The long term objective of our project is to elucidate the molecular mechanisms of neuronal-glial differentiation in the mammalian nervous system. For the short term project (present application for 5 years), we will continue to study the coordinate regulation of two glial proteins [S100 and glial fibrillary acidic (GFAP) proteins] using a set of cell lines derived from a rat peripheral neurotumor RT4. There are four different cell types in the RT4 family. One of the cell types (multipotential stem-cell type) gives rise to three distinct derivative cell types. The stem-cell type expresses only neuronal properties (neuronal-cell type), and the third derivative cell type expresses only glial properties (glial-cell type). Expression of both S100P and GFAP (glial proteins) is mediated by dibutyryl cAMP, but we have observed some differences in cAMP responsiveness between the two proteins. Our results so far obtained indicate that at least one of the glial properties (expression of S100P) which segregates among the derivative cell types is regulated by a negative factor which recognizes a cis-acting element in the 5' flanking region of the S100P gene. In addition, an enhancer-like element is likely to exist in the first intron of the S100P gene. We have isolated S100P and GFAP genomic sequences which include 5' flanking regions and have constructed S100P expression vectors with chloramphenicol acetyltransferase (CAT). We plan to study the phenomenon of glial-specific gene expression by constructing GFACAT expression vectors identifying the DNA sequence elements of S100P and GFAP recognized by coordinate regulator proteins, isolating regulator proteins that bind to the elements and their corresponding genes, and studying the control mechanisms of the regulator genes themselves. We will also study inductional aspects of glial properties by identifying cAMP responsive elements and their binding proteins. The projects in this application deal with mostly molecular biological and biochemical studies. We have already worked out several background technical conditions for this line of work, i.e., (a) cloning and sequencing of the genomic 5'flanking regions of S100P and GFAP, (b) construction of an expression vector with CAT and a series of 5'-deletion mutants for S100 protein, (c) partial purification of nuclear proteins of our cell lines, and (d) gel retardation assays for DNA binding proteins. These studies will contribute to the understanding of the mechanisms of neuronal-glial differentiation and will also shed some light on the mechanisms of cell differentiation and embryological development in general.